OBJECTIVETo investigate the biological function of RIG-G gene by establishing a cell line stably expressing RIG-G protein.

METHODSEctopic RIG-G gene was transfected into U937 cells by using Tet-off expression system. Changes before and after RIG-G expression were detected for cell growth, cell morphology, cell surface antigen and cell cycle regulating proteins.

RESULTSRIG-G protein arrested the cells at G0/G1 phase and inhibited cell growth by increasing the cell cycle inhibitors P21 and P27. As compared to that in control group, the proportion of cells at G0/G1 phase in RIG-G-expressing cell group increased from (43.9 +/- 5.6)% to (63.9 +/- 2.3)% (P < 0.01). The rate of growth inhibition was (68.7 +/- 0.2)%. In addition, an increase in CD11C expression [(61.3 +/- 1.1)% vs. (18.0 +/- 0.4)% (P < 0.01)] and in cells with morphologic features of partial differentiation (smaller cell size, reduced nucleus-cytoplasm ratio, notched nucleus and coarse chromatin) was also observed in RIG-G-expressing cells.

CONCLUSIONSRIG-G has potential abilities to inhibit cell proliferation and promote cell differentiation.

Cell Cycle ; genetics ; Cell Differentiation ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; physiology ; Plasmids ; genetics ; Transfection ; U937 Cells

OBJECTIVETo study the effects of arsenic trioxide (As(2)O(3)) on cell cycle and expression of cyclin dependent kinase inhibitors (CDKIs) in multiple myeloma (MM) cells, and explore its pharmacological mechanism.

METHODSThe DNA content of MM cells line HS-Sultan was analyzed by flow cytometry after exposure to As(2)O(3), the effects on expression of CDKI P15, P16 AND P21 were studied by reverse transcriptase PCR.

RESULTSDNA flow cytometric analysis showed that As(2)O(3) induced most of HS-Sultan cells, arrest at G(0)/G(1) phase and a small fraction at G(2)/M phase and apoptosis occurred mainly in S phase. There was no expression of P15 and P16 mRNA in untreated HS-Sultan cells and 1.0 micromol/L As(2)O(3) could make them expressed after exposed 24 or 48 hours respectively. Expression of P12 mRNA was obviously elevated by As(2)O(3) comparing with that of control.

CONCLUSIONOne of the pharmacological mechanisms of As(2)O(3) is to activate the expression of CDKI P15, P16 and P21, and consequently affect cell proliferation cycle.

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Cycle ; drug effects ; physiology ; Cyclin-Dependent Kinase Inhibitor p15 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p16 ; biosynthesis ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Humans ; Multiple Myeloma ; drug therapy ; metabolism ; pathology ; Oxides ; pharmacology ; RNA, Messenger ; genetics ; Tumor Cells, Cultured

OBJECTIVECytotoxin-associated protein (CagA) of H. pylori has been confirmed to be closely associated with gastric inflammation and tumorigenesis, but the mechanism behind it is little understood. In this study, we try to determine roles of CagA(+) strain in activating PI3K/Akt1 signaling pathway, and affecting expression of p21(WAF1/CIP1) and p27(KIP1), and also in releasing IL-8 in host cells.

METHODSAkt1 phosphorylation and IL-8 levels of CagA(+) and CagA⁻ strain infected AGS cells were detected by ELISAs. Two quantitative RT-PCRs were established to measure p21(WAF1/CIP1) and p27(KIP1) mRNA levels in the CagA(+) and CagA⁻ strain infected cells. LY294002, an inhibitor of PI3K/Akt pathway, was used to define effect of the pathway in IL-8 release.

RESULTSCagA(+) strain could induce an obvious elevation of Akt1 phosphorylation in the infected AGS cells while CagA? strain failed to do so. The CagA(+) H. pylori strain infected AGS cells showed significant drops both in p21(WAF1/CIP1) and p27(KIP1) mRNA levels, whereas the CagA⁻ H. pylori strain caused a remarkable increase in p21(WAF1/CIP1) mRNA without affecting p27(KIP1) gene transcription in the AGS cells. Both the CagA(+) and CagA⁻ H. pylori strains enabled AGS cells to produce close elevated levels of IL-8, and the LY294002 block resulted in unexpected elevations of IL-8 levels.

CONCLUSIONSCagA can activate PI3K/Akt1 pathway that plays an inhibitory role in IL-8 release in H. pylori infected AGS cells. Activation of PI3K/Akt1 pathway and subsequent negative regulation of p21(WAF1/CIP1) and p27(KIP1) expression might be involved in CagA-associated carcinogenesis.

Antigens, Bacterial ; genetics ; physiology ; Bacterial Proteins ; genetics ; physiology ; Cell Line ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; Cyclin-Dependent Kinase Inhibitor p27 ; Gastric Mucosa ; cytology ; enzymology ; microbiology ; Helicobacter pylori ; metabolism ; pathogenicity ; physiology ; Humans ; Interleukin-8 ; secretion ; Intracellular Signaling Peptides and Proteins ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; Transcription, Genetic ; Virulence

OBJECTIVETo study effects of the expression of transforming growth factor (TGF)-beta1 on the growth of Smad4-null pancreatic cancer cells.

METHODSTGF-beta1 eukaryotic expression vector was transfected into pancreatic cancer cell line BxPC3. Effects of the expressison of TGF-beta1 was studied by growth curve analysis and flow cytometry. Cell motility was monitored by wound-healing assay. Western blot was used to estimate the expression level of p21(WAF/CLIP1), a cyclin-dependent kinase inhibitor.

RESULTSTransfection of TGF-beta1 changed the morphology of BxPC3 into spindle shaped cells. The growth rate of BxPC3 began to decrease after the fourth day of TGF-beta1 transfection, compared with the control groups. Flow cytometry showed that the percentages of cells in the S phase were (27.53 +/- 0.02)%, (26.32 +/- 0.01)% and (17.01 +/- 0.03)% in naïve BxPC3, vector-control group and TGF-beta1 transfection group respectively. Lesser cells entered the S phase after TGF-beta1 transfection (P < 0.01), but no difference was seen between the BxPC3 and vector groups (P > 0.05). The expression of p21(WAF/CLIP1) increased upon the expression of TGF-beta1.

CONCLUSIONThe Smad4-independent pathway of TGF-beta1 not only induces epithelial-mesenchymal transition in pancreatic cancer BxPC3, but also inhibits its growth through the up-regulation of p21(WAF/CLIP1).

Cell Line, Tumor ; Cell Movement ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Gene Deletion ; Genetic Vectors ; Humans ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; S Phase ; Smad4 Protein ; genetics ; Transfection ; Transforming Growth Factor beta1 ; biosynthesis ; genetics ; physiology ; Up-Regulation

OBJECTIVETo observe the inhibitory effect of cyclin-dependent kinase inhibitor p21 on regulation of survivin transcription in human liver cancer HepG cells, and explore the related mechanisms.

METHODSDoxorubicin (DOX) was used to treat HepG cells. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG cells by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21, p53 and survivin were detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to determine the cell cycle phases, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 or p300.

RESULTSAfter treatment with DOX, the expression of p53 and p21 was increased, whereas that of survivin was reduced during 24 hours of the treatment. After transfection the p21 level was 2100.1-fold or 980.9-fold enhanced in comparison with that in HepG2 cells or HepG2-pEGFP cells. Survivin level was markedly down-regulated to 0.5% or 0.6% relative to that in the other two groups, nevertheless, significant p53 changes were not observed. Overexpression of p21 resulted in G1/G0 phase arrest (F = 31.59, P < 0.01), meanwhile, E2F-1 mRNA or p300 mRNA were less expressed compared with that in the other controls (F(E2F-1) = 125.28, P < 0.05; Fp300 = 46.01, P < 0.01).

CONCLUSIONp21 could be a potential mediator of survivin suppression at transcription level in HepG2 cells, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.

Antibiotics, Antineoplastic ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; metabolism ; physiology ; Doxorubicin ; pharmacology ; E2F1 Transcription Factor ; genetics ; metabolism ; G1 Phase ; Gene Expression Regulation, Neoplastic ; Hep G2 Cells ; Humans ; Inhibitor of Apoptosis Proteins ; Microtubule-Associated Proteins ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Resting Phase, Cell Cycle ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; p300-CBP Transcription Factors ; genetics ; metabolism

In order to study the potential effects of exogenous WT1 gene isoform on apoptosis in leukemia cell line NB4 and its possible molecular mechanisms, the eukaryotic expression recombinant vector (pCB6(+)/WTA) containing full-length human WT1 isoform (WTA: -17aa/-KTS) cDNA and the vacant vector-alone were introduced into the leukemia cell line NB4 respectively by electroporation. The WTA mRNA and protein in cells were detected by RT-PCR and Western blot. Binding of Annexin V were tested by flow cytometry and agarose gel electrophoresis to verify whether exogenous WTA could induce apoptosis of NB4 cells. Expressions of p21, p53, bcl-2, bcl-XL and c-myc genes were determined by semi-quantitative RT-PCR after introducing recombinant vectors into the NB4 cells. The results showed that in exposure to As(2)O(3) at 0.8 micromol/L for 48 hours, the NB4/WTA cells exhibited the morphological hallmarks of apoptosis, the marked DNA ladder shown by gel electrophoresis, and the enhanced apoptosis rate marked by Annexin V. RT-PCR showed an increase in p21 and c-myc genes expression, a decrease in bcl-2 and a relative constant expression of p53, bcl-XL in NB4/WTA cells. It is concluded that the introduction and expression of exogenous WTA gene can lead to apoptosis of NB4/WTA cells by down-regulating the Bcl-2 gene expression and up-regulating the p21 and c-myc genes expression.
Apoptosis ; genetics ; physiology ; Blotting, Western ; Cell Line, Tumor ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Tumor Suppressor Protein p53 ; genetics ; WT1 Proteins ; genetics ; metabolism ; physiology ; bcl-X Protein ; genetics

BACKGROUNDTo investigate the relationship among human papillomavirus (HPV)16 infection and the expression of telomerase catalytic protein subunit (hTERT), tumor suppressor gene p21waf1, proliferation antigen Ki67 in cervical intraepithelial neoplasias (CIN) and squamous cell carcinomas (SCC) of cervix uteri and their significance.

METHODSTissue microarray combined with in situ hybridization (ISH) and immunohistochemical staining (EliVision plus method) was used to detect the expression of HPV16 RNA, hTERT, Ki67 and p21waf1 proteins in the cervix uteri specimens from 130 subjects, including normal cervical tissue (n=26), CIN (n=46) and SCC (n=58).

RESULTS(1) The positive rate of HPV16 hybridization signals and expression of hTERT, Ki67 in CINII-III, in situ carcinoma and invasive SCC were all significantly higher than in normal cervical tissue (P < 0.05 for all), and was also higher in SCC than in CIN (P < 0.05 for all). There was a significant difference among CIN I, II and III (P < 0.05 for all). (2) The positive expression of p21waf1 protein only in SCC was significantly lower than in normal cervical tissue (P < 0.05), but there was no significant differences among other groups (all P > 0.05). (3) The positive rate of HPV16 and the expression of Ki67 showed respectively positive being correlated with the expression of hTERT (P < 0.05, r=0.339; P < 0.05, r=0.398); HPV16, hTERT and Ki67 showed respectively negative correlation with the expression of p21waf1 (P < 0.05, r=-0.337; P < 0.05, r=-0.248; P < 0.05, r=-0.446); There was no significant relation between HPV16 and Ki67 (P > 0.05).

CONCLUSIONThe results suggest that in tissues of CIN and SCC changes in hTERT, p21waf1 and Ki67 expression may be associated with HPV16 infection and they interact with each other, which can influent the progression of CIN and carcinogenesis of SCC. These biomarkers may be analyzed comprehensively to reveal the detailed mechanism by which HPV16 participate in malignant transformation and to provide some informations on the diagnosis of patients with high risk for malignant progression. Tissue microarray is an efficient platform for high-throughput analysis of genes and their expression products in investigations.

Carcinoma, Squamous Cell ; genetics ; metabolism ; virology ; Cervical Intraepithelial Neoplasia ; genetics ; metabolism ; virology ; Cyclin-Dependent Kinase Inhibitor p21 ; biosynthesis ; genetics ; Female ; Gene Expression Regulation, Neoplastic ; Host-Pathogen Interactions ; Human papillomavirus 16 ; physiology ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Ki-67 Antigen ; biosynthesis ; genetics ; Middle Aged ; Papillomavirus Infections ; genetics ; metabolism ; virology ; Peptide Fragments ; biosynthesis ; genetics ; Telomerase ; biosynthesis ; genetics ; Tissue Array Analysis ; Uterine Cervical Neoplasms ; genetics ; metabolism ; virology

Alternative Splicing ; Apoptosis ; Cell Cycle ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Hep G2 Cells ; Humans ; Inhibitor of Growth Protein 1 ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; physiology ; Nuclear Proteins ; genetics ; metabolism ; physiology ; Plasmids ; Protein Isoforms ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Transfection ; Tumor Suppressor Protein p53 ; metabolism ; Tumor Suppressor Proteins ; genetics ; metabolism ; physiology ; bcl-2-Associated X Protein ; metabolism

WT1 gene encodes a zinc finger transcription factor that regulates transcription of its downstream genes. Some of target genes for WT1 are involved in regulating both cell cycle and cellular proliferation and differentiation. However, WT1 itself is regulated by its upstream genes such as NF-kappaB and GATA-1. Thus there exists a pathway of transcriptional regulation mediated by WT1, which controls development of hematopoietic system. Leukemia results from disrupting the homeostasis among hematopoietic proliferation, differentiation and apoptosis, which is often the consequence of an inappropriate expression of transcription factors and subsequent disruption of the normal gene expression pattern. This article reviews the relationship between the WT1-mediated pathway of transcriptional regulation and leukemia.
Animals ; Carrier Proteins ; genetics ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; DNA-Binding Proteins ; metabolism ; Erythroid-Specific DNA-Binding Factors ; GATA1 Transcription Factor ; Gene Expression Regulation ; Humans ; Leukemia ; etiology ; genetics ; NF-kappa B ; metabolism ; Nuclear Proteins ; genetics ; Retinoblastoma-Binding Protein 7 ; Transcription Factors ; metabolism ; Transcription, Genetic ; WT1 Proteins ; physiology

OBJECTIVETo verify the binding of p53 to p21WAF1/CIP1 gene promoter and detect its binding to hsp90 beta gene promoter in vivo.

METHODSChromatin immunoprecipitation and PCR analysis were used to measure specific gene regulation sequence and Western blot analysis to investigate p53 protein.

RESULTSThe p53 binding sequences on the promoters of p21WAF1/CIP1 and hsp90 beta gene were found in the p53 antibody immunoprecipitated DNA fragments and p53 was detected in the immunoprecipitated samples.

CONCLUSIONSp53 binds to promoters of p21WAF1/CIP1 and hsp90 beta gene in vivo, and regulates the expression of the two genes.

Binding Sites ; Chromatin ; genetics ; isolation & purification ; Cyclin-Dependent Kinase Inhibitor p21 ; Cyclins ; genetics ; metabolism ; Gene Expression Regulation ; HSP90 Heat-Shock Proteins ; genetics ; metabolism ; Humans ; Polymerase Chain Reaction ; methods ; Precipitin Tests ; methods ; Promoter Regions, Genetic ; Protein Binding ; Transcription, Genetic ; Tumor Suppressor Protein p53 ; genetics ; metabolism ; physiology